The present invention relates generally to genetic polymorphisms that are associated with neuropsychiatric disorders and associated conditions including hypothyroidism.
Over 2000 human pathological syndromes are known to result from DNA polymorphisms including insertions, deletions, multiplications and nucleotide substitutions. Finding genetic polymorphisms in individuals and following these variations in families provides a means to confirm clinical diagnoses and to diagnose both predispositions and disease states in carriers, preclinical and subclinical affected individuals. Counseling based upon accurate diagnoses allows patients to make informed decisions about potential parenting, ongoing pregnancy, and early intervention in affected individuals.
Polymorphisms associated with pathological syndromes are highly variable and, consequently, can be difficult to identify. Further, normal polymorphic nucleotide changes can complicate detection of abnormal alleles with changes at different nucleotides. Because multiple alleles within genes are common, one must distinguish disease syndrome-related alleles from neutral (non-disease-related) polymorphisms. Most alleles result from neutral polymorphisms that produce indistinguishable, normally active gene products or express normally variable characteristics like eye color. In contrast, some polymorphic alleles are associated with clinical diseases such as sickle cell anemia. Moreover, the structure of disease-related polymorphisms are highly variable and may result from a single point mutation such as occurs in sickle cell anemia, or from the expansion of nucleotide repeats as occurs in fragile X syndrome and Huntington""s chorea.
Once a polymorphism or region of interest is identified, a wide variety of technologies exist which may be employed in the diagnosis of heritable syndromes. Traditionally, the diagnosis of such syndromes relied upon enzyme activity testing, statistical analysis, or invasive diagnostic procedures. Recent advances in DNA and related technologies including restriction fragment length polymorphism (RFLP) analysis, the polymerase chain reaction (PCR) and monoclonal or polyclonal antibody based assays provide additional rapid and highly accurate methods to screen for the presence of polymorphisms associated with heritable pathologies.
Among the different polymorphisms, the presence of unstable DNA sequences, such as the expansion of simple nucleotide sequence repeats in genomic DNA, has recently been implicated as a mechanism leading to a number of genetic disorders including pathologies associated with neuropsychiatric disorders such as mental retardation. Mental retardation (MR), which can be generally defined as a slowness or developmental impairment associated with adaptive behavior, is a prominent feature of many neurodevelopmental syndromes. MR is a lifelong disability that can place extreme demands on the families and on the health care system in general. Information obtained from the Incidence and Prevalence Database estimates that there are 6 million persons in the U.S. (3% of the population) with mental retardation. MR can be categorized as mild mental retardation (MMR, IQ 50-70) or as severe mental retardation (SMR, IQ less than 50). It is noted that SMR can be further subcategorized. An average SMR prevalence rate per 1000 people is thought to be as follows: ages 0-4, 1.7; ages 5-9, 2.8; ages 10-14, 3.6; ages 15-19, 4.0; ages 20-29, 3.8; ages 30-39, 3.3; 40-49, 2; ages 50-59, 1.2; ages over 60, 1.0. In 1977, nearly 150,000 adults with mental retardation were institutional residents; by 1992 their numbers had declined by 48% to just under 78,000.
The diagnosis of SMR does not usually occur in the first few years of life, rather it is usually identified later, typically at the school age years. The explanation why there is decreasing prevalence rates in the older age has been attributed to a higher than average mortality among the severely mentally retarded and possibly due to errors in the method used in gathering the data. Approximately one-half of the MR studies have shown rates of MR to be gender-specific. For SMR, the male-to-female ratio, there has been observed a 20% excess of males, which is thought to be due to sex-linked genetic factors.
Due to the prevalence of mental retardation (MR) and its pattern of heritability, the identification of chromosomal regions that are associated with MR pathologies has been the focus of significant research. Mental retardation affects approximately 1% of the U.S. population with mutations in the X-chromosome estimated to cause between 30-50% of these cases (Glass, I. A., (1991) X linked mental retardation. J Med Genet 28:361-371). The genetic mechanisms involved in a number of these X-linked syndromes have been identified and include repetitive DNA expansion in Fragile X (Verkerk, et al., xe2x80x9cIdentification of a gene (FMR-1) containing a CGG repeat coincident with a fragile X breakpoint cluster region exhibiting length variation in fragile X syndromexe2x80x9d, Cell 65:905 (1991)) and FRAXE (Gecz, et al., xe2x80x9cIdentification of the gene FMR2, associated with FRAXE mental retardationxe2x80x9d, Nat Genet 13:105-108 (1996)), microdeletions (Billuart, et al., xe2x80x9cIdentification by STS PCR screening of a microdeletion in Xp21.3-22.1 associated with non-specific mental retardationxe2x80x9d, Hum Mol Genet 5:977-979 (1996)), and point mutations in the Mental retardation, Aphasia, Shuffling gait, and Adducted thumbs (MASA) syndrome (Schrander-Stumpel, et al., xe2x80x9cSpectrum of X-linked hydrocephalus (HSAS), MASA syndrome, and complicated spastic paraplegia (SPG1): clinical review with six additional familiesxe2x80x9d, Am J Med Genet 57:107-116 (1995)) and Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus syndrome (CRASH) (Fransen, et al., xe2x80x9cCRASH syndrome: clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single genexe2x80x9d, Eur J Hum Genet 3:273-284 (1995)). While this research has provided significant insight into these X-linked syndromes, the causes of the majority of MR remain idiopathic at the current time.
The identification and characterization of specific polymorphisms associated with heritable syndromes such as MR are crucial components in the design of informative diagnostic assays. By identifying specific regions in the human genome which contain disease related polymorphisms, statistical analysis of the prevalence and penetrance of the syndrome is possible. Further, as different formulas are utilized for the assessment of autosomal recessive, autosomal dominant, and X-linked genetic diseases, the identification of the chromosomal location of the polymorphism is a crucial factor in the assessment of pedigree related risk analysis. Such information allows accurate risk assessments to take into account 1) the number of different alleles at each gene locus, 2) the relative frequency of each allele in the population (the most informative have more than one common allele), and 3) whether alleles are distributed randomly throughout the population. As technologies for assessing the presence or absence of a specific polymorphism or polymorphic region are well developed, the primary limitation on diagnostic assays is the lack of information on polymorphisms associated with different pathologies.
What is needed in the art is the identification of novel regions in the human genome which contain polymorphisms that are associated with heritable syndromes. The identification of such regions is particularly useful in that it allows for the design of informative assays and diagnostic tests for susceptibility factors associated with the occurrence of such syndromes. The existence of informative assays which test for the presence of such heritable factors allows the accurate diagnosis of affected individuals and provides these individuals and health care professionals with the knowledge necessary to make informed decisions based on the presence or absence of a disease-associated polymorphism.
The present invention is directed to the discovery that polymorphisms in the q13 region of the X chromosome are associated with non-Fragile X mental retardation, hypothyroidism and a number of neuropsychiatric disorders including depression, bipolar affective disorder, attention-deficit/hyperactivity disorder (ADHD), and a psychotic disorder. Polymorphisms in this region, designated xe2x80x9cPCTG4xe2x80x9d, were identified by genotyping a large sample of DNAs from a diverse population of mentally ill individuals with respect to a number of loci. These polymorphisms are found to have an increased prevalence in non-Fragile X males and females with mental retardation, autism, depression, hypothyroidism, attention-deficit/hyperactivity disorder (ADHD), and/or a psychotic disorder. The present invention relates to the utilization of the polymorphic regions disclosed herein in the diagnosis and assessment of mental retardation. Since such pathologies can now be detected earlier (i.e., before overt symptoms appear) and more definitively, better treatment options will be available in those individuals identified as having pathologies associated with the disclosed polymorphisms.
The PCTG4 region is shown by fluorescence in situ hybridization to be localized to Xq13 and to span more than 55 kb. A number of polymorphic regions exhibiting base pair insertions, base pair deletions, and repetitive nucleotide sequences associated with neuropsychiatric disorders in the PCTG4 region within Xq13 are identified herein. One of these polymorphisms consists of a 12 base pair insertion in the coding region of the HOPA gene. Another polymorphism consists of a 15 base pair deletion between 6 and 7 base pairs upstream from where the 12 base pair polymorphism occurs. An additional polymorphism consists of a pentanucleotide repeat approximately 7 kb upstream of the 12 base pair polymorphism. Another polymorphism consists of a dinucleotide repeat approximately 4.5 kb downstream of the 12 base pair polymorphism.
In one embodiment, the invention provides isolated nucleic acid molecules which encode PCTG4 region polymorphisms. Isolated nucleic acid can include PCTG4 region polymorphisms having the sequences identified in Table 1 or having sequences that are complementary to these nucleic acid sequences, preferentially hybridize to them and remain stably bound to them under at least moderate, and optionally, under high stringency conditions. In another embodiment, the invention provides a vector comprising polymorphic PCTG4 region sequences. A recombinant cell comprising such a vector inserted into a host cell is also provided. In another embodiment, the invention provides a polypeptide such as an antibody capable of specifically binding a polymorphic epitope on a polypeptide encoded by a gene in the PCTG4 region, for example a HOPA or neuroligin-3 polypeptide. Optionally, the antibody is a monoclonal antibody. In yet another embodiment, the invention provides animals having PCTG4 region transgenes.
In other embodiments, the invention provides methods for screening for PCTG4 region polymorphisms. In one embodiment, the invention provides a method for screening for a polymorphism associated with mental retardation in a subject by determining the presence of a polymorphism in the subject""s PCTG4 nucleic acid sequence obtained from the subject, wherein the polymorphism associated with mental retardation is characterized by an insertion or repetitive nucleotide units. In a specific embodiment of this method the polymorphism is the PCTG4 12 base pair insert polymorphism, the 15 base pair deletion polymorphism, the PCTG4 dinucleotide repeat polymorphism, or the PCTG4 pentanucleotide repeat polymorphism disclosed herein. In a more specific embodiment of the invention, the presence of a polymorphism in the PCTG4 nucleic acid sequence is determined by a differential nucleic acid analysis technique such as restriction fragment length polymorphism analysis, direct sequence analysis or polymerase chain reaction analysis.
In another embodiment, the invention provides a method for identifying a patient""s susceptibility to pathologies associated with mental retardation by determining the patient""s PCTG4 polymorphism pattern, comparing it to the wild type PCTG4 pattern, and then looking for differences indicative of a susceptibility to pathologies associated with mental retardation. In a related embodiment, the invention provides a method of identifying a polymorphism associated with mental retardation by comparing a PCTG4 gene sequence isolated from a mentally retarded subject to a known wild-type PCTG4 gene sequence and identifying recurrent polymorphisms. Typically, a PCTG4 gene sequence used in such a comparison is the HOPA gene, the neuroligin-3 gene, or both the HOPA gene and the neuroligin-3 gene. In specific embodiments of these methods, the presence of a polymorphism in PCTG4 nucleic acid sequences is determined by a differential nucleic acid analysis technique such as restriction fragment length polymorphism analysis, direct sequence analysis, DNA chip analysis, or polymerase chain reaction analysis.
Other embodiments of the invention include kits and articles of manufacture for use in the methods disclosed herein as well as cell based assays for assessing the effects of candidate agents on the activity of genes from the PCTG4 region.